Phytases, a specific group of monoester phosphatases, are required to initiate the release of phosphate (“P”) from phytate (myo-inositol hexophosphate), the major storage form of P in cereal foods or feeds (Reddy, N. R. et al., “Phytates in Legumes and Cereals,” Advances in Food Research, 28:1 (1982)). Because simple-stomached animals like swine and poultry as well as humans have little phytase activity in their gastrointestinal tracts, nearly all of the ingested phytate P is indigestible. This results in the need for supplementation of inorganic P, an expensive and non-renewable nutrient, in diets for these animals. More undesirably, the unutilized phytate-P excreted through manure of these animals becomes P pollution of the environment (Cromwell, G. L. et al., “P- A Key Essential Nutrient, Yet a Possible Major Pollutant—Its Central Role in Animal Nutrition,” Biotechnology In the Feed Industry; Proceedings Alltech 7th Annual Symposium, p. 133 (1991)). Furthermore, phytate chelates with essential trace elements like zinc and produces nutrient deficiencies such as growth and mental retardation in children ingesting mainly plant origin foods without removal of phytate.
Two phytases, phyA and phyB, from Aspergillus niger NRRL3135 have been cloned and sequenced (Ehrlich, K. C. et al., “Identification and Cloning of a Second Phytase Gene (phys) from Aspergillus niger (ficuum),” Biochem. Biophys. Res. Commun., 195:53–57 (1993); Piddington, C. S. et al., “The Cloning and Sequencing of the Genes Encoding Phytase (phy) and pH 2.5-optimum Acid Phosphatase (aph) from Aspergillus niger var. awamori,” Gene 133:56–62 (1993)). Recently, new phytase genes have been isolated from Aspergillus terreus and Myceliophthora thermophila (Mitchell et al., “The Phytase Subfamily of Histidine Acid Phosphatases: Isolation of Genes for Two Novel Phytases From the Fungi Aspergillus terreus and Myceliophthora thermophila,” Microbiology 143:245–52, (1997)), Aspergillus fumigatus (Pasamontes et al., “Gene Cloning, Purification, and Characterization of a Heat-Stable Phytase from the Fungus Aspergillus fumigatus” Appl. Environ. Microbiol., 63:1696–700 (1997)), Emericella nidulans and Talaromyces thermophilus (Pasamontes et al., “Cloning of the Phytase from Emericella nidulans and the Thermophilic Fungus Talaromyces thernophilus,” Biochim. Biophys. Acta. 1353:217–23 (1997)), and maize (Maugenest et al., “Cloning and Characterization of a cDNA Encoding a Maize Seedling Phytase,” Biochem. J. 322:511–17 (1997)).
Various types of phytase enzymes have been isolated and/or purified from Enterobacter sp. 4 (Yoon et al., “Isolation and Identification of Phytase-Producing Bacterium, Enterobacter sp. 4, and Enzymatic Properties of Phytase Enzyme.,” Enzyme and Microbial Technology 18:449–54 (1996)), Klebsiella terrigena (Greiner et al., “Purification and Characterization of a Phytase from Klebsiella terrigena,” Arch. Biochem. Biophys. 341:201–06 (1997)), and Bacillus sp. DS11 (Kim et al., “Purification and Properties of a Thermostable Phytase from Bacillus sp. DS11,” Enzyme and Microbial Technology 22:2–7 (1998)). Properties of these enzyme have been studied. In addition, the crystal structure of phyA from Aspergillus ficuum has been reported (Kostrewa et al., “Crystal Structure of Phytase from Aspergillus ficuum at 2.5 A Resolution,” Nature Structure Biology 4:185–90 (1997)).
Hartingsveldt et al. introduced phyA gene into A. niger and obtained a ten-fold increase of phytase activity compared to the wild type. (“Cloning, Characterization and Overexpression of the Phytase-Encoding Gene (phyA) of Aspergillus Niger,” Gene 127:87–94 (1993)). Supplemental microbial phytase of this source in the diets for pigs and poultry has been shown to be effective in improving utilization of phytate-P and zinc (Simons et al., “Improvement of Phosphorus Availability By Microbial Phytase in Broilers and Pigs,” Br. J. Nutr. 64:525 (1990); Lei, X. G. et al., “Supplementing Corn-Soybean Meal Diets With Microbial Phytase Linearly Improves Phytate P Utilization by Weaning Pigs,” J. Anim. Sci., 71:3359 (1993); Lei, X. G. et al., “Supplementing Corn-Soybean Meal Diets With Microbial Phytase Maximizes Phytate P Utilization by Weaning Pigs,” J. Anim. Sci. 71:3368 (1993); Cromwell, G. L. et al., “P- A Key Essential Nutrient, Yet a Possible Major Pollutant—Its Central Role in Animal Nutrition,” Biotechnology In the Feed Industry; Proceedings Alltech 7th Annual Symposium, p. 133 (1991)). However, the cost of the limited commercial phytase supply and its instability when subjected to heat during feed pelleting preclude its practical use in animal industry (Jongbloed, A. W. et al., “Effect of Pelleting Mixed Feeds on Phytase Activity and Apparent Absorbability of Phosphorus and Calcium in Pigs,” Animal Feed Science and Technology, 28:233–42 (1990)). Moreover, phytase produced from A. niger is presumably not the safest source for human food manufacturing.
Thus, there is a need to improve phytase production for application by the food and feed industry.